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The International Journal of... Aug 2023The forces that cells, tissues, and organisms exert on the surface of a soft substrate can be measured using Traction Force Microscopy (TFM), an important and... (Review)
Review
The forces that cells, tissues, and organisms exert on the surface of a soft substrate can be measured using Traction Force Microscopy (TFM), an important and well-established technique in Mechanobiology. The usual TFM technique (two-dimensional, 2D TFM) treats only the in-plane component of the traction forces and omits the out-of-plane forces at the substrate interfaces (2.5D) that turn out to be important in many biological processes such as tissue migration and tumour invasion. Here, we review the imaging, material, and analytical tools to perform "2.5D TFM" and explain how they are different from 2D TFM. Challenges in 2.5D TFM arise primarily from the need to work with a lower imaging resolution in the z-direction, track fiducial markers in three-dimensions, and reliably and efficiently reconstruct mechanical stress from substrate deformation fields. We also discuss how 2.5D TFM can be used to image, map, and understand the complete force vectors in various important biological events of various length-scales happening at two-dimensional interfaces, including focal adhesions forces, cell diapedesis across tissue monolayers, the formation of three-dimensional tissue structures, and the locomotion of large multicellular organisms. We close with future perspectives including the use of new materials, imaging and machine learning techniques to continuously improve the 2.5D TFM in terms of imaging resolution, speed, and faithfulness of the force reconstruction procedure.
Topics: Microscopy, Atomic Force; Traction; Mechanical Phenomena; Focal Adhesions; Stress, Mechanical; Cell Adhesion
PubMed: 37290687
DOI: 10.1016/j.biocel.2023.106432 -
Fluids and Barriers of the CNS Jun 2023The choroid plexus (ChP) has been suggested as an alternative central nervous system (CNS) entry site for CCR6 Th17 cells during the initiation of experimental...
The choroid plexus (ChP) has been suggested as an alternative central nervous system (CNS) entry site for CCR6 Th17 cells during the initiation of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). To advance our understanding of the importance of the ChP in orchestrating CNS immune cell entry during neuroinflammation, we here directly compared the accumulation of CD45 immune cell subsets in the ChP, the brain and spinal cord at different stages of EAE by flow cytometry. We found that the ChP harbors high numbers of CD45 resident innate but also of CD45 adaptive immune cell subsets including CCR6 Th17 cells. With the exception to tissue-resident myeloid cells and B cells, numbers of CD45 immune cells and specifically of CD4 T cells increased in the ChP prior to EAE onset and remained elevated while declining in brain and spinal cord during chronic disease. Increased numbers of ChP immune cells preceded their increase in the cerebrospinal fluid (CSF). Th17 but also other CD4 effector T-cell subsets could migrate from the basolateral to the apical side of the blood-cerebrospinal fluid barrier (BCSFB) in vitro, however, diapedesis of effector Th cells including that of Th17 cells did not require interaction of CCR6 with BCSFB derived CCL20. Our data underscore the important role of the ChP as CNS immune cell entry site in the context of autoimmune neuroinflammation.
Topics: Animals; Mice; Encephalomyelitis, Autoimmune, Experimental; Choroid Plexus; Neuroinflammatory Diseases; Brain; Central Nervous System; Mice, Inbred C57BL
PubMed: 37264368
DOI: 10.1186/s12987-023-00441-4 -
Antigen recognition detains CD8 T cells at the blood-brain barrier and contributes to its breakdown.Nature Communications May 2023Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of...
Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of CD8 T cells are found in MS lesions, and antigen (Ag) presentation at the BBB has been proposed to promote CD8 T cell entry into the CNS. Here, we show that brain endothelial cells process and cross-present Ag, leading to effector CD8 T cell differentiation. Under physiological flow in vitro, endothelial Ag presentation prevented CD8 T cell crawling and diapedesis resulting in brain endothelial cell apoptosis and BBB breakdown. Brain endothelial Ag presentation in vivo was limited due to Ag uptake by CNS-resident macrophages but still reduced motility of Ag-specific CD8 T cells within CNS microvessels. MHC class I-restricted Ag presentation at the BBB during neuroinflammation thus prohibits CD8 T cell entry into the CNS and triggers CD8 T cell-mediated focal BBB breakdown.
Topics: Humans; Blood-Brain Barrier; CD8-Positive T-Lymphocytes; Endothelial Cells; Central Nervous System; Multiple Sclerosis; Histocompatibility Antigens Class I
PubMed: 37253744
DOI: 10.1038/s41467-023-38703-2 -
Archives of Pharmacal Research Jun 2023GPR35, an orphan receptor, has been waiting for its ligand since its cloning in 1998. Many endogenous and exogenous molecules have been suggested to act as agonists of... (Review)
Review
GPR35, an orphan receptor, has been waiting for its ligand since its cloning in 1998. Many endogenous and exogenous molecules have been suggested to act as agonists of GPR35 including kynurenic acid, zaprinast, lysophosphatidic acid, and CXCL17. However, complex and controversial responses to ligands among species have become a huge hurdle in the development of therapeutics in addition to the orphan state. Recently, a serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), is reported to be a high potency ligand for GPR35 by investigating the increased expression of GPR35 in neutrophils. In addition, a transgenic knock-in mouse line is developed, in which GPR35 was replaced with a human ortholog, making it possible not only to overcome the different selectivity of agonists among species but also to conduct therapeutic experiments on human GPR35 in mouse models. In the present article, I review the recent advances and prospective therapeutic directions in GPR35 research. Especially, I'd like to draw attention of readers to the finding of 5-HIAA as a ligand of GPR35 and lead to apply the 5-HIAA and human GPR35 knock-in mice to their research fields in a variety of pathophysiological conditions.
Topics: Mice; Humans; Animals; Hydroxyindoleacetic Acid; Ligands; Serotonin; Receptors, G-Protein-Coupled; Kynurenic Acid
PubMed: 37227682
DOI: 10.1007/s12272-023-01449-y -
Cell Reports May 2023Monocytes are abundant immune cells that infiltrate inflamed organs. However, the majority of monocyte studies focus on circulating cells, rather than those in tissue....
Monocytes are abundant immune cells that infiltrate inflamed organs. However, the majority of monocyte studies focus on circulating cells, rather than those in tissue. Here, we identify and characterize an intravascular synovial monocyte population resembling circulating non-classical monocytes and an extravascular tissue-resident monocyte-lineage cell (TR-MC) population distinct in surface marker and transcriptional profile from circulating monocytes, dendritic cells, and tissue macrophages that are conserved in rheumatoid arthritis (RA) patients. TR-MCs are independent of NR4A1 and CCR2, long lived, and embryonically derived. TR-MCs undergo increased proliferation and reverse diapedesis dependent on LFA1 in response to arthrogenic stimuli and are required for the development of RA-like disease. Moreover, pathways that are activated in TR-MCs at the peak of arthritis overlap with those that are downregulated in LFA1 TR-MCs. These findings show a facet of mononuclear cell biology that could be imperative to understanding tissue-resident myeloid cell function in RA.
Topics: Humans; Monocytes; Synovial Membrane; Arthritis, Rheumatoid; Inflammation
PubMed: 37204925
DOI: 10.1016/j.celrep.2023.112513 -
Microbiology Spectrum Jun 2023Porphyromonas gingivalis is an important periodontal pathogen that can cause vascular injury and invade local tissues through the blood circulation, and its ability to...
Porphyromonas gingivalis is an important periodontal pathogen that can cause vascular injury and invade local tissues through the blood circulation, and its ability to evade leukocyte killing is critical to its distal colonization and survival. Transendothelial migration (TEM) is a series of that enable leukocytes to squeeze through endothelial barriers and migrate into local tissues to perform immune functions. Several studies have shown that P. gingivalis-mediated endothelial damage initiates a series of proinflammatory signals that promote leukocyte adhesion. However, whether P. gingivalis is involved in TEM and thus influences immune cell recruitment remains unknown. In our study, we found that P. gingivalis gingipains could increase vascular permeability and promote Escherichia coli penetration by downregulating platelet/endothelial cell adhesion molecule 1 (PECAM-1) expression . Furthermore, we demonstrated that although P. gingivalis infection promoted monocyte adhesion, the TEM capacity of monocytes was substantially impaired, which might be due to the reduced CD99 and CD99L2 expression on gingipain-stimulated endothelial cells and leukocytes. Mechanistically, gingipains mediate CD99 and CD99L2 downregulation, possibly through the inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In addition, our model confirmed the role of P. gingivalis in promoting vascular permeability and bacterial colonization in the liver, kidney, spleen, and lung and in downregulating PECAM-1, CD99, and CD99L2 expression in endothelial cells and leukocytes. P. gingivalis is associated with a variety of systemic diseases and colonizes in distal locations in the body. Here, we found that P. gingivalis gingipains degrade PECAM-1 to promote bacterial penetration while simultaneously reducing leukocyte TEM capacity. A similar phenomenon was also observed in a mouse model. These findings established P. gingivalis gingipains as the key virulence factor in modulating the permeability of the vascular barrier and TEM processes, which may provide a new rationale for the distal colonization of P. gingivalis and its associated systemic diseases.
Topics: Mice; Animals; Gingipain Cysteine Endopeptidases; Porphyromonas gingivalis; Platelet Endothelial Cell Adhesion Molecule-1; Transendothelial and Transepithelial Migration; Endothelial Cells; Phosphatidylinositol 3-Kinases; Adhesins, Bacterial
PubMed: 37199607
DOI: 10.1128/spectrum.04769-22 -
Pharmaceuticals (Basel, Switzerland) Apr 2023Unfractionated heparin has multiple pharmacological activities beyond anticoagulation. These anti-inflammatory, anti-microbial, and mucoactive activities are shared in... (Review)
Review
Unfractionated heparin has multiple pharmacological activities beyond anticoagulation. These anti-inflammatory, anti-microbial, and mucoactive activities are shared in part by low molecular weight and non-anticoagulant heparin derivatives. Anti-inflammatory activities include inhibition of chemokine activity and cytokine synthesis, inhibitory effects on the mechanisms of adhesion and diapedesis involved in neutrophil recruitment, inhibition of heparanase activity, inhibition of the proteases of the coagulation and complement cascades, inhibition of neutrophil elastase activity, neutralisation of toxic basic histones, and inhibition of HMGB1 activity. This review considers the potential for heparin and its derivatives to treat inflammatory lung disease, including COVID-19, ALI, ARDS, cystic fibrosis, asthma, and COPD via the inhaled route.
PubMed: 37111341
DOI: 10.3390/ph16040584 -
Current Protocols Apr 2023Inflammation is the body's response to injury and harmful stimuli and contributes to a range of infectious and noninfectious diseases. Inflammation occurs through a...
Inflammation is the body's response to injury and harmful stimuli and contributes to a range of infectious and noninfectious diseases. Inflammation occurs through a series of well-defined leukocyte-endothelial cell interactions, including rolling, activation, adhesion, transmigration, and subsequent migration through the extracellular matrix. Being able to visualize the stages of inflammation is important for a better understanding of its role in diseases processes. Detailed in this article are protocols for imaging immune cell infiltration and transendothelial migration in vascular tissue beds, including those in the mouse ear, cremaster muscle, brain, lung, and retina. Also described are protocols for inducing inflammation and quantifying leukocytes with FIJI imaging software. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of croton oil dermatitis Alternate Protocol 1: Induction of croton oil dermatitis using genetically fluorescent mice Basic Protocol 2: Intravital microscopy of the mouse cremaster muscle Support Protocol: Making a silicone stage Basic Protocol 3: Wide-field microscopy of the mouse brain Basic Protocol 4: Imaging the lungs (ex vivo) Alternate Protocol 2: Inflating the lungs without tracheostomy Basic Protocol 5: Inducing, imaging, and quantifying infiltration of leukocytes in mouse retina.
Topics: Mice; Animals; Transendothelial and Transepithelial Migration; Croton Oil; Leukocytes; Inflammation; Dermatitis
PubMed: 37078364
DOI: 10.1002/cpz1.739 -
Advanced Drug Delivery Reviews Jun 2023In the presence of tissue inflammation, injury, or cancer, myeloid cells are recruited to disease regions through a multi-step process involving myelopoiesis,... (Review)
Review
In the presence of tissue inflammation, injury, or cancer, myeloid cells are recruited to disease regions through a multi-step process involving myelopoiesis, chemotaxis, cell migration, and diapedesis. As an emerging drug delivery approach, cell-mediated drug delivery takes advantage of the cell recruitment process to enhance the active transport of therapeutic cargo to disease regions. In the past few decades, a variety of nano-engineering methods have emerged to enhance interactions of nanoparticles with cells of interest, which can be adapted for cell-mediated drug delivery. Moreover, the drug delivery field can benefit from the recent clinical success of cell-based therapies, which created cell-engineering methods to engineer circulating leukocytes as 'living drug delivery vehicles' to target diseased tissues. In this review, we first provide an overview of myeloid cell recruitment and discuss how various factors within this process may affect cell-mediated delivery. In the second part of this review article, we summarize the status quo of nano-engineering and cell-engineering approaches and discuss how these engineering approaches can be adapted for cell-mediated delivery. Finally, we discuss future directions of this field, pointing out key challenges in the clinical translation of cell-mediated drug delivery.
Topics: Humans; Drug Carriers; Nanomedicine; Drug Delivery Systems; Nanoparticles; Myeloid Cells; Cell- and Tissue-Based Therapy
PubMed: 37068659
DOI: 10.1016/j.addr.2023.114827 -
Advanced Science (Weinheim,... Jun 2023Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the...
Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push-pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.
Topics: Transendothelial and Transepithelial Migration; Endothelial Cells; Endothelium; Actins; Mechanical Phenomena; Neoplasms
PubMed: 37051804
DOI: 10.1002/advs.202206554